AMPK Methods and Protocols

3.2 Cell Preparation
and Transfection

1. Cultured cells: Maintain cultured cells (e.g., mouse embryonic
   fibroblasts, HeLa cervical cancer cells, Cos7 African green
   monkey fibroblasts, etc.), at 37Cin5%CO 2 at 90% relative
   humidity in the appropriate cell culture medium (seeNote 11).


2. Pre-warm cell culture media, Opti-MEM, and trypsin at 37C.


3. Prepare the transfection mixture in a 1.5 ml sterile microcen-
   trifuge tube. For FuGENE HD transfection, mix 2 μgof
   plasmid DNA encoding the desired osABKAR and 6μlof
   FuGENE HD in 100μl Opti-MEM. If multiple constructs
   are transfected together, the ratio should be optimized by
   testing the expression of each construct. Transfection reagents
   are not limited to FuGENE HD, and other transfection
   reagents (e.g., Lipofectamine) can be used instead. If resorting
   to a different transfection reagent, the protocol must be mod-
   ified according to the manufacturer’s instruction.


4. Incubate the transfection mixture for 20 min at room temper-
   ature (seeNote 12).


5. In the meantime, prepare glass cover slips coated with poly-D-
   lysine. Store cover slips in individual wells in a 6-well plate (see
   Note 13).


6. Trypsinize cells, transfer 210
   104 number of cells into a
   15 ml tube, and spin them down at 362
   gfor 3 min (see
   Note 14).


7. Resuspend the cells in 10 ml of cell culture medium.


8. Add 500μl of resuspended cells to 100μl of the transfection
   mixture, and mix them well by tapping.


9. Add 80μl of the cell suspension onto each cover slip.


10. For cell adhesion, leave the cover slips in the incubator for 2 h.
    Duration of the incubation should be adjusted for individual
    cell types.


11. Add 2 ml of fresh cell culture medium to each well.


12. Acquire images 36–48 h after transfection (seeNote 15).

3.3 Live Cell Imaging Monitoring AMPK activity with ABKAR/osABKARs is performed
as follows:

1. Replace culture medium with fresh culture medium 2 h before
   live cell imaging (seeNote 16).


2. Place a cover slip into a metal frame filled with 450μl of the
   imaging medium (seeNote 17).


3. Use a fluorescence microscope for image acquisition. Consis-
   tent temperature and CO 2 conditions are desirable irrespective
   of the duration of the imaging session (seeNotes 18and 19 ).

Genetically-Encoded Sensors and Impeders 263